Accep ed Manusc ip
E alua ion o HPCD ba ch ea men s on enzyme inac i a ion kine ics and selec ed
quali y cha ac e is ics o cloudy juice om Golden delicious apples
A.E. Ille a, M.T. Sanz, S. Bel án, R. Melgosa, A.G. Solaesa, M.O. Ruiz
PII: S0260-8774(17)30448-X
DOI: 10.1016/j.j oodeng.2017.10.017
Re e ence: JFOE 9050
To appea in: Jou nal o Food Enginee ing
Please ci e his a icle as: A.E. Ille a, M.T. Sanz, S. Bel án, R. Melgosa, A.G. Solaesa, M.O.
Ruiz, E alua ion o HPCD ba ch ea men s on enzyme inac i a ion kine ics and selec ed quali y
cha ac e is ics o cloudy juice om Golden delicious apples, Jou nal o Food Enginee ing (2017), doi:
10.1016/j.j oodeng.2017.10.017
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HPCD inac i a ion a e o PPO and PME inc eased wi h inc easing p and T.
Sensi i i y o empe a u e change is simila o HPCD and mild he mal ea men .
PME is mo e HPCD esis an han PPO.
HPCD ea men dec eased pa icle size o cloudy apple juice.
Colloidal s abili y and nu i ional p ope ies we e no a ec ed by HPCD.
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E alua ion o HPCD ba ch ea men s on enzyme inac i a ion kine ics and selec ed
1
quali y cha ac e is ics o cloudy juice om Golden delicious apples
2
3
A.E. Ille a, M. T. Sanz
∗
, S. Bel án, R. Melgosa. A. G. Solaesa, M. O. Ruiz
4
Depa men o Bio echnology and Food Science (Chemical Enginee ing Sec ion),
5
Uni e si y o Bu gos, 09001 Bu gos. Spain
6
7
Abs ac
8
Cloudy apple juice has been ea ed by high p essu e ca bon dioxide (HPCD) as non- he mal
9
echnology o inac i e polyphenoloxidase and pec inme hyles e ase in ba ch mode. S i ing
10
speed ( om 200 o 600 pm) induced an inc ease in he enzyme inac i a ion a e while a iple
11
cycle o p essu iza ion/dep essu iza ion led o he same enzyme inac i a ion e iciency. Enzyme
12
inac i a ion kine ics we e de e mined a di e en empe a u es ( om 35 o 45 ºC) and p essu es
13
( om 10 o 20 MPa). Da a we e desc ibed by he i s o de kine ic model and he Weibull
14
model. Fo he i s o de kine ic model, decimal educ ion ime o HPCD ea men was ound
15
o be smalle han o mild hea ing, in he same empe a u e ange. The same endency was
16
obse ed o he i s decimal educ ion ime in he Weibull model. HPCD ea men esul ed in a
17
homogeniza ion e ec e lec ed in he shi ing o he pa icle size dis ibu ion owa ds smalle
18
diame e s a e ea men . HPCD ea men did no esul in a change o wa e and oxala e
19
soluble pec in con en , o al phenolic compounds and hid oxyme hyl u u al con en .
20
21
22
∗ Co esponding au ho . Tel.: +34 947 258810. Fax: ++34947258831. E-mail add ess e [email protected]
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Keywo ds: Cloudy apple juice, HPCD, inac i a ion kine ics pa ame e s, p ope ies
23
1. In oduc ion
24
Consump ion o cloudy apple juice seems o be mo e bene icial han consuming clea apple juice
25
due o changes in phenolic compounds and loss o pec ins du ing cla i ica ion p ocess
26
(Ma kowski e al., 2015). Howe e , colo and cloud s abili y a e main challenges in p ese a ion
27
o cloudy apple juice du ing i s p ocessing and s o age. Enzyma ic b owning in cloudy apple
28
juice is due o he ac ion o polyphenol oxidase (PPO) ha ca alyzes he oxida ion o phenolic
29
compounds o quinones. On he o he hand, he mechanism o cloud s abili y is no ye
30
unde s ood. Among he di e en mechanisms p oposed, elec os a ic epulsion by nega i e
31
cha ges p esen in he pa ly deme hyla ed pec in due o galac u onic esidues could be
32
esponsible o cloud s abili y a oiding agg ega ion (Zhao e al., 2008). In his ega d, he ac i i y
33
o pec inme hyles e ase (PME) causes pec in deme hyla ion ha could p ecipi a e wi h calcium
34
ions p esen in he juice causing cla i ica ion and he loss o u bidi y. Howe e , o he
35
componen s wi h nega i e cha ge ha e been also epo ed in he li e a u e as s abilizing agen s o
36
he cloud in he se um such as phospha ide acids o cloud lipids (K ap enbaue e al., 2006).
37
The mal ea men s ha e been adi ionally used o he inac i a ion o mic oo ganisms and
38
enzymes ha a ec nega i ely he quali y o oods. Howe e , hese me hods cause undesi able
39
e ec s in quali y a ibu es such as la o and loss o nu i ional componen s. High p essu e
40
ca bon dioxide (HPCD) ea men has been p oposed as an al e na i e non- he mal pas eu iza ion
41
echnique ha can inac i a e ce ain mic oo ganisms and enzymes unde mild ope a ion
42
condi ions. Typically, ope a ing p essu e does no exceed 50 MPa and empe a u e anges
43
be ween 20 and 50 ºC, below pas eu iza ion empe a u e (Manzocco e al., 2016). The
44
mechanism o p essu ized CO
2
on enzyme inac i a ion is no ye ully unde s ood. In he
45
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li e a u e di e en app oaches can be ound such as pH lowe ing due o dissol ed CO
2
,
46
con o ma ional changes o o ma ion o di e en complex wi h CO
2
molecules (Hu e al., 2013).
47
Some s udies ha e been ound in he li e a u e ega ding he e ec o HPCD on enzymes o
48
cloudy apple juice om Fuji apples. Niu e al. (2010) e alua ed he quali y o cloudy Fuji apple
49
juice p ocessed om apple slices p e iously ea ed by HPCD. These au ho s ound ha PPO
50
could be comple ely inac i a ed a 20 MPa o 20 min and a empe a u e as low as 25 ºC;
51
howe e , esidual ac i i y o 18% was s ill ound o PME a e ea men a 65 ºC and 20 MPa
52
o 20 min. Gui a al. (2007) ound highe alues o esidual ac i i y han he wo k o Niu e al.
53
(2010), o PPO in HPCD- ea ed cloudy Fuji apple juice in a ba ch mode (40% a 30 MPa and
54
55 ºC o 60 min). When using a con inuous HPCD sys em, holding imes o 10 min we e
55
needed a 22 MPa and 60 ºC o achie e comple e inac i a ion o PPO bu , a he same wo king
56
condi ions, s ill PME esidual ac i i y o 42% was ob ained (Xu e al., 2011).
57
F om p e ious epo ed esul s, i can be clea ly obse ed ha he e is a g ea a ie y in he
58
li e a u e da a on enzyme inac i a ion om cloudy apple. This is a main d awback in he
59
comme cializa ion o his echnology and eliable da a on juice quali y a e s ill needed.
60
Acco ding o Wang e al. (2010) he in luence o agi a ion on enzyme inac i a ion unde high-
61
p essu e en i onmen is a key ac o in he design o eac o s o indus ial scale olume.
62
Howe e , he e ec o s i ing speed on enzyme ac i i y has no been conside ed in mos o he
63
s udies. Wang e al. (2010) de e mined he e ec o agi a ion a 40ºC on isoamylase, ob ained
64
om a e men a ion b o h om Pseudomonas amylode amosa a e cell emo al, and a
65
comme cial β-amylase unde supe c i ical ca bon dioxide, a di e en ope a ing imes (up o 120
66
min) and di e en ope a ing p essu e ( om 11.0 o 17.2 MPa), obse ing ha he ac i i y o bo h
67
enzymes dec eased by inc easing he speed o agi a ion.
68

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In his wo k, he e ec o HPCD ea men on some quali y cha ac e is ics o cloudy apple juice
69
om Golden delicious has been s udied. The e ec o some a iables, non-p e iously s udied in
70
li e a u e, on enzyme inac i a ion om cloudy apple juice, has been conside ed such as he e ec
71
o s i ing speed and he numbe o successi e p essu iza ion/dep essu iza ion cycles. Enzyme
72
inac i a ion kine ic da a pa ame e s a e also necessa y o scale-up HPCD p ocess. This ega d,
73
enzyme inac i a ion kine ics o PPO and PME ha e been de e mined a di e en ope a ing
74
p essu es and empe a u es. Enzyme inac i a ion kine ic da a ha e been desc ibed by he i s
75
o de kine ic model and he Weibull model as an al e na i e o he classical i s o de kine ic
76
model. Some o he impo an quali y pa ame e s o cloudy apple juice ha e been also de e mined
77
be o e and igh a e HPCD ea men , such as pec in con en , pa icle size dis ibu ion, colo
78
and an ioxidan capaci y.
79
2. Ma e ials and me hods
80
2.1 Juice p epa a ion
81
Golden delicious apples we e peeled and cu in cubes and hen added o a 0.3 % o L-asco bic
82
acid solu ion o a oid enzyma ic b owning du ing p ocessing. Apple cubes we e wiped and
83
immedia ely squeezed wi h a sc ew juice ex ac o . The liqueu was il e ed wi h 2 laye s o
84
cheeseclo h. The pH o he juice ob ained was 3.89 ± 0.01 and he soluble solid con en was 12.5
85
± 0.1 °B ix. The juice was s o ed ozen a −18 °C un il u he use.
86
2.2 HPCD equipmen and p ocessing
87
The HPCD cell was made o s ainless s eel (SS-316) and had an in e nal olume o 100 mL wi h
88
a maximum ope a ing p essu e and empe a u e o 30 MPa and 80 ºC, espec i ely (Melgosa e
89
al., 2017). Fo each expe imen , 40 mL o apple juice we e cha ged in o he high p essu e cell
90
ha was imme sed in a he mos a ic wa e ba h a he ope a ing empe a u e. The magne ic
91
s i ing was hen connec ed and he sys em was p essu ized by a using a sy inge pump wi h a
92
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p essu e con olle (ISCO 260 D) up o he desi ed p essu e. CO
2
was ed in o he high p essu e
93
cell h ough a sin e ed s ainless s eel mic o- il e wi h a po e size o 10 µm (B iongos e al.,
94
2016). This ega d, Ishikawa e al. (1995) showed ha he concen a ion o CO
2
in he sample
95
was in luenced by he way CO
2
was ed in o he sample, inc easing by dec easing he po e size
96
o he il e a 25 MPa and 35ºC.
97
Samples we e collec ed pe iodically o ollow he inac i a ion kine ics o he enzymes.
98
Expe imen s we e ca ied ou in he anges o empe a u e (T) om 35 o 45 ºC, p essu e (p)
99
om 10 o 20 MPa and s i ing speed om 200 o 600 pm. The e ec o he sampling p ocedu e
100
and he numbe o p essu iza ion/dep essu iza ion cycles on enzyme ac i i y was also analyzed.
101
To compa e he e ec o he HPCD ea men on enzyme ac i i y wi h mild hea ing ea men ,
102
cloudy apple juice was hea ed a a mosphe ic p essu e in he same empe a u e ange ( om 35 o
103
45 ºC). Samples we e aken pe iodically a di e en ea men imes up o 120 min.
104
Some o he impo an quali y a ibu es o cloudy apple juice, such as pa icle size dis ibu ion, ξ
105
po en ial, u bidi y, pH, pec in con en , o al phenolic compounds, non-enzyma ic b owning and
106
an ioxidan capaci y we e also de e mined be o e and igh a e HPCD ea men .
107
2.3 Physico-chemical analysis
108
2.3.1. De e mina ion o PPO ac i i y
109
The ac i i y o PPO was de e mined spec opho ome ically by using a 0.05 M ca echol (Sigma
110
Ald ich) solu ion p epa ed in a 0.1 M phospha e bu e (pH 6.5) as subs a e. Samples we e
111
analyzed by adding 100 µL o apple juice in o 2.9 mL subs a e solu ion. Oxida ion o ca echol
112
was de e mined immedia ely by he inc ease in abso bance a 420 nm by using a Jasco V-750
113
spec opho ome e equipped wi h a Pel ie he mos a ed cell holde and a wa e pump o keep he
114
empe a u e cons an a 30 ºC. The PPO ac i i y was aken as he e y i s linea pa o he
115
eac ion cu e.
116
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2.3.2. De e mina ion o PME ac i i y
117
PME ac i i y was de e mined by using an au oma ic i a o sys em (Me ohm  Ti ando) by
118
using a 1 % o pec in solu ion (Al a Aesa  pec in ci us) p epa ed in NaCl 0.3 M as subs a e.
119
50 mL o pec in solu ion was mixed wi h 1 mL o cloudy apple juice and pH was adjus ed o 7.5
120
wi h NaOH 0.02 N. Du ing pec in hyd olysis a 30 ºC, pH was main ained a 7.5 by adding
121
NaOH 0.02 N. The amoun o NaOH added o 15 min was eco ded. One PME ac i i y uni
122
(UPE) is de ined as he mic omoles o ca boxylic g oups p oduced pe minu e and mL o juice a
123
pH 7.5 and 30 ºC (B iongos e al., 2016)
.
124
Rela i e esidual ac i i ies o PPO and PME we e e alua ed as:
125
Residualac i i y,RA= !"#
$%&'%·100%=,,
-
·100 [1]
126
2.3.3. De e mina ion o pec ic subs ances
127
Pec ic subs ances in cloudy apple juice be o e and a e HPCD ea men ha e been de e mined
128
acco ding o Robe son (1979) by p og essi e ex ac ion o he alcohol insoluble solids by wa e
129
(high me hoxyl pec ins a e ex ac ed) and ammonium oxala e (low me hoxyl pec ins a e
130
ex ac ed). Wa e soluble pec ins play an impo an ole in he u bidi y o he cloudy apple
131
juices ac ing as colloid s abilize s, while oxala e-soluble ac ion can o m gels wi h poly alen
132
me al ions (Robe son, 1979). Pec ic subs ances a e de e mined spec opho ome ically a
133
520 nm a e o al hyd olysis o galac u onic acids by using m-hyd oxydiphenil (MHDP) as a
134
ch omogenic eagen . Solu ions we e eshly p epa ed be o e analysis: 0.15% solu ion o m-
135
hyd oxydiphenyl (Sigma Ald ich) in 0.5% NaOH and 0.0125 M sodium e abo a e (Pan eac) in
136
concen a ed sulphu ic acid. Fo pec in de e mina ion, 1 mL o wa e ex ac and 5 mL o he
137
sodium e abo a e solu ion we e mixed and placed in a wa e -ice ba h. The mix u e is hen
138
hea ed in wa e ba h a 80 ºC o 6 min and subsequen ly cooled again in a wa e -ice ba h.
139
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0.1 mL o he m-hyd oxydiphenyl solu ion was added and a e homogeniza ion o he mix u e,
140
abso bance a 520 nm was measu ed as a unc ion o ime. Acco ding o Iba z e al. (Iba z e al.,
141
2006) spec opho ome ic da a measu emen s we e aken a he highes abso bance ins ead o a a
142
ce ain eac ion ime. A calib a ion cu e was p epa ed wi h s anda d solu ions o D-galac u onic
143
acid (Sigma Ald ich) by ollowing he same colo ime ic me hod. A blank was also p epa ed
144
wi h no D-galac u onic acid.
145
2.3.4. Pa icle size dis ibu ion and ze a-po en ial
146
Pa icle size dis ibu ion (PSD) was de e mined by lase di ac ion wi h a Mas e size 2000
147
(Mal e n® Ins ., MA). The sys em uses a lase ligh a 750 nm wa eleng h o size pa icles om
148
0.4 o 2000 µm by ligh di ac ion. Pa icle size dis ibu ion was calcula ed by he F aunho e
149
model. Size dis ibu ions ( olume ac ions agains pa icle size) be o e and a e HPCD
150
ea men we e calcula ed and he weigh -a e age sizes exp essed as:
151
• The equi alen su ace a ea mean diame e : D/3,22=∑nd5
6∑n
⁄d5
8 [2]
152
• The equi alen olume mean diame e : D/4,32=∑nd5
:∑n
⁄d5
6 [3]
153
whe e d
lc
is he diame e o he pa icle and n
c
is he pe cen age o pa icles.
154
To desc ibe dis ibu ion wid h one common pa ame e is he Span:
155
Span=#
=,>.@
A#
=,>.B
#
=,>.C
[4]
156
whe e D
,0.9
, D
,0.1
and D
0.5
a e he pa icle size bellow which, 90%, 50% and 10% o he
157
pa icles lies.
158
ζ-po en ial was de e mined wi h a Ze asize Nano ZS appa a us, using he Lase Dopple
159
Velocime y echniques. Samples we e dilu ed 1:50 wi h deionized wa e and il e ed h ough
160
5 µm il e . ζ-po en ial gi es an indica ion o he po en ial s abili y o a colloidal sys em and i is
161
a good index o he colloidal elec os a ic epulsi e o ces. Pa icles will epel each o he when
162
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li e a u e abou he e ec o he numbe o cycles on enzyme inac i a ion by HPCD on esh
291
juices.
292
3.3 E ec o mild hea ing on PPO inac i a ion
293
PPO inac i a ion kine ics we e de e mined a a mosphe ic p essu e in he empe a u e ange
294
om 35 o 45 ºC (same empe a u e ange as o HPCD ea men , Figu e 2). The maximum
295
educ ion o PPO ac i i y was nea ly 40 % a e 2 h o hea ing a 45 ºC indica ing ha PPO o
296
cloudy Golden delicious apple juice was a he s able unde mild empe a u es. Simila
297
inac i a ion deg ees o PPO om cloudy Fuji apple juice we e ob ained by Gui e al. (2007).
298
A e 1 hou o mild hea ing, hese au ho s eached a maximum educ ion o PPO ac i i y o
299
20% a 45 ºC. Howe e , Niu e al. (2010) ound ha when subjec ing apple slices o mild hea ing
300
ea men om 25 o 45 ºC o 20 min and hen p ocessing hem in o cloudy apple juice, PPO
301
ac i i y inc eased by 22-51%. Buckow e al. (2009) also ound ha a 45 ºC, PPO ac i i y om
302
cloudy apple juice inc eased 30%, a ibu ing his beha io o he elease o la en PPO.
303
Enzyme inac i a ion kine ic da a unde mild hea ing condi ions and a a mosphe ic p essu e we e
304
desc ibed by he i s -o de model. Pa ame e s a e collec ed in Table 1. Inac i a ion a e
305
cons an , k, inc eased wi h he ope a ing empe a u e, he e o e he co esponding D alues
306
dec eased wi h empe a u e. The empe a u e sensi i i y pa ame e , z
T
, and he ac i a ion ene gy
307
we e e alua ed as 36 ºC and 53 kJ/mol, espec i ely (see Table 1). Values o he same o de
308
we e epo ed by Gui e al. (2007) o cloudy Fuji apple juice, 27.0 ºC o z
T
and 72.0 kJ/mol, o
309
Ea. The mal s abili y o ex ac ed apple PPO om Golden delicious was s udied in he
310
empe a u e ange om 68 o 78 ºC by Yemenicigly e al. (1997). These au ho s epo ed a z
T
311
alue o 9.9 ºC. The highe alue ob ained in his wo k o z
T
in he empe a u e ange om 35 o
312
45 ºC migh indica e a lowe suscep ibili y o empe a u e change a empe a u es lowe han he
313

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one obse ed a pas eu iza ion empe a u es. In any case, he alues o he ac i a ion ene gy
314
we e simila in bo h empe a u e anges.
315
3.4. E ec o HPCD ea men on PPO inac i a ion
316
Enzyme inac i a ion kine ics o PPO o cloudy Golden deliciuos apple juice ha e been
317
de e mined a 10 MPa in he empe a u e ange om 35 o 45 ºC (Figu e 3a). By inc easing
318
empe a u e, enzyme inac i a ion a e also inc eased. In addi ion o he in insic e ec o
319
empe a u e on enzyme inac i a ion, by inc easing empe a u e, al hough CO
2
densi y dec eased,
320
he di usi i y o CO
2
can be imp o ed and accele a e he molecula collisions be ween CO
2
321
molecules and he enzyme. Figu e 3b shows he inac i a ion kine ics a 45 ºC in he p essu e
322
ange om 10 o 20 MPa. Highe ope a ing p essu es led o as e inac i a ion a es, bu he
323
ac ion o he esis an o m o he enzyme was nea ly independen o he ope a ing p essu e,
324
being a ound 6 %. This alue was lowe han he minimal esidual ac i i y o PPO om cloudy
325
Fuji apple juice epo ed by Gui e al. (2007), 37%, a 30 MPa and 55 ºC o 60 min. In any case,
326
compa ison wi h o he HPCD sys ems is di icul o es ablish since he inac i a ion eached
327
would depend no only on he ype and sou ce o he enzyme and he expe imen al condi ions
328
such as p essu e, empe a u e and ea men ime bu also on he agi a ion o he sys em and he
329
a io sample olume/cell olume since his las pa ame e de e mines he amoun o CO
2
injec ed
330
o he sys em. Un o una ely in mos o he s udies hese pa ame e s a e no de ailed. PPO om
331
cloudy Fuji apple juice was comple ely inac i a ed by a con inuous HPCD sys em a 22 MPa and
332
60 ºC o 10 min (Xu e al., 2011). This could sugges a highe e iciency o a con inuous sys em
333
due o he u bulen ene gy, compa able wi h a good agi a ion in a discon inuous sys em.
334
Howe e , 60 ºC canno be conside ed as non- he mal and esidual ac i i y as high as 47% was
335
ob ained in he con inuous sys em a 43 ºC and 25 MPa o 2 min. (Xu e al., 2011).
336
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Inac i a ion kine ic da a by HPCD we e i ed o he i s o de kine ic model and he Weibull
337
model (Table 2). Al hough he Weibull model yielded he bes i ing quali y, he i s o de
338
kine ic model desc ibed he inac i a ion cu e qui e well and he e o e, kine ic pa ame e s o
339
he linea eg ession model ha e been also lis ed in Table 2 o compa ison wi h da a epo ed in
340
he li e a u e.
341
Fo he i s o de kine ic model, inac i a ion a e cons an , k, inc eased wi h empe a u e and
342
p essu e. Consequen ly, D alues dec eased wi h inc easing p essu e and empe a u e. A each
343
ope a ing empe a u e, D alues we e smalle o HPCD ea men (Table 2) han he
344
co esponding alues o mild hea ing ea men (Table 1) p o ing ha HPCD ea men was
345
mo e e ec i e han he mal ea men alone. Fo example D was equal o 515 min a 45 ºC and
346
a mosphe ic p essu e and dec eased o 104 min a 45 ºC and 10 MPa.
347
z
T
and ac i a ion ene gy alues we e de e mined as 29 ºC and 64 kJ/mol, espec i ely, o he
348
i s o de kine ic model (see Table 2). z
T
and ac i a ion ene gy alues we e o he same o de as
349
hose lis ed o mild hea ing ea men (Table 1). This mean ha he sensi i i y o empe a u e
350
change was simila o HPCD as o mild hea ing ea men in he empe a u e ange co e ed in
351
his wo k. The same endency in z
T
and E
a
was ound by Liu e al. (2008) in he inac i a ion o
352
PPO o ed bee ex ac s. These au ho s ound ha unde mild hea ing ea men a a mosphe ic
353
p essu e (35-55 ºC), z
T
and E
a
we e equal o 34 ºC and 57 kJ/mol, espec i ely. A e HPCD
354
ea men a 37.5 MPa, in he same empe a u e ange, hey ob ained alues o 36 ºC and
355
54 kJ/mol o z
T
and E
a
, espec i ely. Howe e , Gui e al. (2007) ound o PPO o cloudy Fuji
356
apple juice ha z
T
inc eased om 27 o 108 ºC and ac i a ion ene gy dec eased om 72 o
357
18 kJ/mol unde mild hea ing ea men (35-55 ºC) and HPCD a 30 MPa in he same
358
empe a u e ange, espec i ely. This inc ease in z
T
and dec ease in Ea ound by Gui e al. (2007)
359
would indica e ha he enzyme inac i a ion become less empe a u e dependen unde
360
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p essu ized CO
2
. This beha io is di icul o unde s and and i has no been obse ed in ou
361
wo k. As i has been p e iously men ioned, in addi ion o he in insic e ec o empe a u e on
362
enzyme inac i a ion, empe a u e has wo di e en e ec s on impo an p ope ies o CO
2
. An
363
inc ease in empe a u e in ol es an imp o emen o mass anspo p ope ies enhancing CO
2
364
di usi i y and he numbe o collisions be ween he CO
2
and he enzyme, bu on he o he hand,
365
CO
2
densi y and he e o e CO
2
solubili y in he aqueous medium dec eases. I seems ha hese
366
e ec s play bo h an impo an ole, since simila sensi i i y o empe a u e was ound in mild
367
hea ea men and in HPCD.
368
By using equa ions 8 and 10 z
P
and V
a
ha e been de e mined as 25 MPa and -251 cm
3
/mol,
369
espec i ely o he i s o de kine ic model. The nega i e alue o he ac i a ion olume
370
indica ed ha PPO inac i a ion was a o ed by inc easing ope a ing p essu e. O he alues o
371
ac i a ion olumes epo ed in he li e a u e o PPO inac i a ion om di e en sou ces a e o
372
he same o de , -271.7 and -307.9 cm
3
/mol o he labile and s able ac ion o PPO o wa e
373
melon a 50 ºC in he p essu e ange o 8-30 MPa (Liu e al., 2013) o -94.31 cm
3
/mol o PPO
374
om cloudy Fuji apple juice a 55ºC in he p essu e ange om 8 o 30 MPa (Gui e al., 2007).
375
The Weibull pa ame e s a e also lis ed in Table 2. Scale, α, and shape, β, pa ame e s we e used
376
o calcula e he ime equi e o inac i a e 1 log (
d=1
) o PPO. Simila o he D- alues o he i s
377
o de kine ic model,
d=1
dec eased wi h ope a ing p essu e and empe a u e. As sugges ed by an
378
Boekel (2002) is impo an o de e mine he e ec o empe a u e, and in he p esen wo k o
379
p essu e, on bo h pa ame e s o he Weibull model. Scale pa ame e was s a is ically signi ican
380
dependen on empe a u e and p essu e, when es ed a he 95% signi icance le el o a linea
381
ela ionship, dec easing wi h p essu e and empe a u e. The shape ac o , n, was less han 1
382
excep o 10 MPa and 35 ºC. The conca i y shape o he inac i a ion cu e could be unde s ood
383
as a as e inac i a ion o he labile o m o he enzyme and he p esence o a esis an o m o
384
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he enzyme o HPCD ea men (Figu es 3a and 3b). The shape pa ame e , β, was ound o be
385
s a is ically signi ican dependen on p essu e bu al hough he e seemed o be a end in
386
empe a u e dependence o β, i was no s a is ically signi ican , when es ed a he 95%
387
signi icance le el o a linea ela ionship. In any case, i mus be highligh ed ha only h ee
388
empe a u es ha e been s udied o de e mine he e ec o empe a u e. Acco ding o an Boekel
389
(2002), he scale pa ame e , α, could be modelled in a simila way o he classical D alue o he
390
i s o de kine ic model, sugges ing a linea dependence o he log α on empe a u e and
391
conside ing ha β did no depend on empe a u e:
392
p{|n=}V−~V [16]
393
A z
T
’ alue can be also de ined:
394
=V

B
[17]
395
The alue o he z
T
’ is lis ed in Table 2, oge he wi h he quali y o he i ing. Al hough he
396
concep o z
T
and z’
T
is di e en since z
T
is ob ained om he linea pa , whe eas z’
T
akes also
397
in o accoun he nonlinea pa (Van Boekel, 2002), simila alues we e ob ained o bo h
398
pa ame e s (z
T
= 29 ºC and z’
T
= 27 ºC).
399
In his wo k, i was also ound a linea dependence o he log α on p essu e:
400
p{|n=}8−~8d [18]
401
Al hough i was ound a s a is ically signi ican linea ela ionship be ween β and p, he in e se
402
o he slope o log α e sus p was also e alua ed. Analogous o z’
T
i was de ined a z’
p
403
=−V

]
[19]
404
This alue is epo ed in Table 2 and qui e simila alues can be obse ed o z
p
and z
p
’.
405
3.5. E ec o HPCD on PME
406
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To compa e he HPCD esis ance o PME and PPO, PME inac i a ion kine ics we e de e mined
407
o he expe imen s pe o med a 45 ºC and 10 MPa and 20 MPa (Figu e 4). PME inac i a ion
408
a e was also de e mined a a mosphe ic p essu e and 45 ºC. A a mosphe ic p essu e, i was
409
ound ha esidual PME ac i i y was s ill 90% a e 120 min a 45ºC (Figu e 4). Teleszko e al.
410
(2016) s udied he mal ea men o di e en apple cul i a s, ounding ha PME om cloudy
411
Golden deliciuous apple juice was one o he apple cul i a s ha p esen ed s onges
412
he mos abili y (30% o esidual ac i i y a e hea ing 5 min a 100 ºC). Niu e al. (2010) ound
413
ha he RA o PME om cloudy Fuji apple juice om apple slices p e iously p ehea ed o
414
20 min a he co esponding wo king empe a u e, a ied be ween 82 o 72 % a 35 and 45ºC,
415
espec i ely, bu wi h e o ba s highe han 10% o RA a each empe a u e (g aphical lec u e).
416
A e HPCD ea men , highe inac i a ion a es we e obse ed han o mild hea ing ea men a
417
he same empe a u e. Al hough, esidual ac i i y was s ill a ound 60 % a 20 MPa and 45 ºC
418
a e 60 min. This indica es ha PME om cloudy Golden delicious apple juice is a he s able o
419
HPCD a ope a ing empe a u es conside ed as non- he mal ea men . Di e en HPCD
420
inac i a ion alues o PME om cloudy apple juice ha e been epo ed in he li e a u e. Niu e
421
al. (2010) epo ed a minimal esidual ac i i y o 18% o PME o cloudy Fuji apple juice om
422
apple slices ea ed by HPCD a 20 MPa and 65 ºC o 20 min. In a con inuous sys em, PME
423
om cloudy Fuji apple juice s ill p esen ed a esidual ac i i y o 40% a 22 MPa and 60ºC o 10
424
min (Xu e al., 2011). HPCD inac i a ion o PME ex ac s om apple juice was ound o be
425
mo e e ec i ely inac i a ed a 30 MPa o 60 min, eaching 5% o esidual ac i i y a 55 ºC (Zhi
426
e al., 2008). Howe e , in his case, esul s a e di e en o compa e since an ex ac ed enzyme
427
suspended in a bu e solu ion can gi e di e en inac i a ion esul s om hose ob ained in an
428
o iginal juice.
429

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Resul s ob ained in his wo k, indica e ha PME om cloudy apple juice was mo e esis an o
430
HPCD han PPO in he p essu e ange co e ed in his wo k. In he li e a u e, as o he mal
431
ea men , he highe s abili y has been a ibu ed o he mo e complex s uc u e o PPO wi h
432
h ee o ou subuni s, in highe plan s, han PME, wi h one subuni . This s uc u e, makes i
433
mo e suscep ible o HPCD ea men (Niu e al., 2010). In addi ion, PME in ui juices is mo e
434
di icul o be e ec i ely inac i a ed, since PME is bound o he plan cell wall, which con ains
435
na u al s abilizing ac o s (Zhou e al., 2010).
436
PME inac i a ion kine ic da a we e desc ibed by he i s o de model a a mosphe ic p essu e
437
(Table 1). The decimal educ ion ime a 45 ºC and a mosphe ic p essu e was ound o be much
438
highe han o PPO a he same condi ions. HPCD inac i a ion da a a 45ºC and 10 MPa and
439
20 MPa we e i ed o he i s o de kine ic model and he Weibull model (Table 2). The
440
Weibull model yielded he bes i ing quali y, bu s ill an accep able good i ing o he i s
441
o de kine ic model was ob ained. Fo he i s o de model, inac i a ion a e cons an inc eased
442
wi h p essu e, bu lowe alues han o PPO inac i a ion we e ob ained and he e o e highe D
443
alues. Rega ding he Weibull model, he scale and shape pa ame e s, bo h, dec eased wi h
444
ope a ing p essu e a 45ºC. These pa ame e s we e used o calcula e he ime needed o inac i a e
445
1 log (
d=1
) o PME a he wo p essu es s udied. The
d=1
calcula ed by he Weibull model is
446
much highe han he co esponding D alues by he i s o de kine ic model. The e o e, in his
447
case, he use o he i s o de kine ic model could lead o an unde p ocessing es ima ion o
448
PME inac i a ion.
449
3.6. E ec o HPCD on di e en quali y pa ame e s o cloudy apple juice
450
Some quali y pa ame e s ha e been de e mined igh a e HPCD ea men a 45 ºC, 20 MPa and
451
60 min o ea men ime.
452
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pH. Ini ial pH o cloudy apple juice was 3.89 ± 0.01 and immedia ely a e HPCD ea men , pH
453
d opped 0.15 uni s o 3.74 ± 0.01. Signi ican di e ences ha e been de e mined among sample
454
pH a e ages be o e and a e HPCD ea men when applying he Fishe ’s leas signi ican
455
me hod a p- alue ≤ 0.05 (Table 3). Gui e al (2007) epo ed a d op o 0.3 uni s om 3.95 o
456
3.66 ± 0.03 o Fuji cloudy apple juice a e HPCD ea men a 22 MPa and 55 ºC o 60 min.
457
Acco ding o Meysammi e al. (1992), juices and be e ages can be conside ed as an aqueous
458
elec oly e sys em o mul iple ions. Unde p essu ized CO
2
a numbe o dissocia ion eac ions
459
can ake place libe a ing some o hem H
+
ions ha could educe he pH o he media. In any
460
case, a e 2 h a e dep essu iza ion pH e u ned o a alue close o i s ini ial alue (pH = 3.87 ±
461
0.01).
462
Colou pa ame e s. Table 3 lis s he L*, a*, b* pa ame e s o cloudy apple juice be o e and
463
a e HPCD ea men . Du ing b owning, he L* alues dec ease, he juice’s colou becomes
464
da ke , while a* and b* alues inc ease, wi h mo e ed and yellow componen s (K ap enbaue e
465
al., 2006). A e HPCD ea men , ligh ness (L*) p esen ed sligh ly, bu signi ican , lowe alues;
466
howe e , 99% o he ligh ness was s ill e ained. No signi ican di e ence was obse ed in he
467
edness componen bu a signi ican inc ease o he yellowness (b*) alue has been obse ed
468
indica ing mo e yellow componen s igh a e HPCD ea men . The change in colou , ∆E (Eq.
469
5) is also p esen ed in Table 3. Sligh ly no iceable di e ences in colo a e HPCD ea men
470
ha e been de e mined, ∆E ≈ 0.51 ± 0.05 (Yuk e al., 2014). O he au ho s ound ha L*, a* and
471
b* alues emained almos cons an a e HPCD ea men a 55 ºC and di e en ope a ing
472
p essu es (8-30 MPa) o 60 min (Gui e al., 2006).
473
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To al polyphenolic compounds. To al polyphenols emained unchanged a e HPCD. Gaspe i
474
e al. (2009) also ound no signi ican di e ences in polyphenols con en a e HPCD ea men
475
o a blend o Golden delicious and G anny Smi h apple a 10 MPa and 36 ºC o 10 min.
476
An ioxidan capaci y. An ioxidan capaci y o Golden cloudy apple juice has been de e mined
477
by he ABTS es s (Table 3). Sligh ly highe alues we e ob ained a e HPCD ea men
478
al hough di e ences be o e and a e ea men we e ound o be no signi ican (p ≤ 0.05).
479
Hyd oxyme hyl u u al. Table 3 p esen s he HMF con en be o e and a e HPCD ea men .
480
F om hese esul s, i can be concluded ha non enzyma ic b owning eac ions we e no
481
signi ican du ing HPCD p ocessing, p obably due o he mild empe a u es employed in HPCD
482
ea men s.
483
Pa icle size dis ibu ion. The pa icle size dis ibu ion o un ea ed cloudy apple juice anged
484
om 0.275 µm o 416.87 µm wi h h ee maxima a 0.63, 19.96 and 208.93 µm (see Figu e 5).
485
A e HPCD ea men , he pa icle ange shi ed o smalle pa icle sizes om 0.04 µm o
486
104.71 µm wi h one main maximum a 0.182 µm. Table 3 lis s alues o D[3,2] and D[4,3] o
487
eshly cloudy apple juice and a e HPCD ea men . The alues a e HPCD ea men we e
488
signi ican ly lowe han hose o eshly cloudy apple juice. The homogeniza ion e ec o HPCD
489
can be also obse ed in he alues o d(0.1), d(0.5) and d(0.9), which co espond o he size o
490
pa icle below which 10%, 50% and 90% o he sample lies, espec i ely. This homogeniza ion
491
e ec induced by HPCD could be a ibu ed o he explosi e ac ion and he bubbling o CO
2
492
om he juice du ing decomp ession (Zhou e al., 2010).
493
Fo eshly cloudy apple juice, he o al olume o all pa icles wi h diame e less han 5 µm
494
ep esen ed 56.5% o he o al pa icles olume, while his numbe inc eased up o 96% a e
495
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HPCD ea men . This ac is also e lec ed in he Span alues (Table 3) ha shows a highe
496
deg ee o uni o mi y o he cloudy apple juice a e HPCD ea men .
497
Tu bidi y. The u bidi y o he cloudy apple juice a e HPCD is conside ably highe han ha o
498
eshly cloudy apple juice (Table 3). I seems ha he e was a co ela ion be ween juice’s
499
u bidi y and pa icle size dis ibu ion Howe e , no co ela ion was ound wi h PME esidual
500
ac i i y since, a e HPCD ea men , PME ac i i y was s ill mo e han 60%.
501
Pec in con en . In his wo k, soluble pec in p esen ed sligh ly highe alues a e HPCD (Table
502
4). HPCD ea men could esul in a mo e dissolu ion o pec in in ui cell in o he juice,
503
inc easing he alue o soluble pec in. In addi ion, acco ding o he PSD p e iously epo ed,
504
HPCD induced an homogeniza ion wi h lowe pa icle size diame e s, which could esul in a
505
highe solubili y o he pec in; since, in he li e a u e, i has been epo ed ha pec in solubili y in
506
wa e inc eases by dec easing i s polyme size (Sila e al., 2009) howe e no signi ican
507
di e ences ha e been ound a p ≤ 0.05. No signi ican di e ences ha e been also de e mined
508
o he oxala e-soluble pec in.
509
In he li e a u e, Zhou e al. (2010) epo ed no change in wa e soluble pec in when ea ing
510
peach juice by HPCD a 30 MPa and 55 ºC a di e en ea men imes, al hough hese au ho s
511
obse ed an inc ease o he la ge pa icle a e HPCD ea men . Howe e , Yu e al (2013)
512
obse ed ha soluble pec in o HPCD- ea ed banana pulp a 20 MPa, du ing 30 min a di e en
513
empe a u es in he ange om 45 o 60 ºC, was signi ican ly lowe han he un ea ed pulp.
514
These au ho s a ibu ed his beha io due o in e ac ion o pec in and p o ein o gelling o pec in
515
ha may occu du ing HPCD ea men (Yu e al., 2013).
516
ξ po en ial. ξ po en ial is an indica ion o colloidal s abili y o he juice. Bo h, un ea ed and
517
ea ed apple juice p esen ed nega i e alues o -22.0 mV and -22.3 mV, espec i ely. These
518
alues we e wi hin he ange epo ed by Co ak and Co edig (2006) o ξ po en ial as a unc ion
519
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Table 1. Kine ic pa ame e s a 0.1 MPa o he i s o de kine ic model o PPO inac i a ion by
657
mild he mal hea ing (35-45ºC) and PME a 45 ºC.
658
PPO Fi s o de kine ic model
T, ºC k, min
-
1
D, min R
2
MRD
35
40
45
0.00102 ± 0.00003
0.00166 ± 0.00005
0.00194 ± 0.00003
980
602
515
0.9814
0.9791
0.9993
1.6
1.1
1.1
z
T
= 36 ± 11 ºC (R
2
= 0.9188)
E
a
= 53 ± 14 kJ/mol (R
2
= 0.9248)
PME, 45ºC 0.00038 ± 0.00003 2632 0.9799 0.8

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Table 2. Kine ic pa ame e s o he Weibull and he i s o de kine ic models o PPO and PME inac i a ion by HPCD. 659
PPO Weibull model Fi s o de kine ic model
p, MPa T, ºC
α
, min β
d=1
, min R
2
MRD k, min
-
1
D, min R
2
MRD
10
10
10
35
40
45
98 ± 2
72 ± 2
41 ± 2
1.13 ± 0.06
0.93 ± 0.04
0.89 ± 0.05
205
177
105
0.9940
0.9939
0.9960
2.3
3.4
4.9
0.0044 ± 0.0001
0.0060 ± 0.0001
0.0096 ± 0.0002
227
167
104
0.9891
0.992
0.992
3.4
3.9
6.8
z’
T
= 27 ± 4 ºC; (R
2
= 0.9726) z
T
= 29 ± 3 ºC; (R
2
= 0.9897)
E
a
= 64 ± 8 kJ/mol; (R
2
= 0.9834)
12.5
15
20
45
45
45
30 ± 1
23.7 ± 0.4
17.2 ± 0.9
0.74 ± 0.02
0.70 ± 0.02
0.59 ± 0.02
93
78
71
0.9983
0.9995
0.9981
2.9
1.2
5.9
0.015 ± 0.001
0.019 ± 0.001
0.026 ± 0.004
67
53
38
0.966
0.953
0.965
23.6
15.6
44.3
z’
P
= 27 ± 3 MPa; (R
2
= 0.9729) z
P
= 25 ± 5 MPa; (R
2
= 0.9226)
Va=- 251 ± 48 cm
3
/mol; (R
2
= 0.9332 )
PME α, min β
d=1
, min R
2
MRD k, min
-1
D, min R
2
MRD
p, MPa T, ºC
10 45 785 ± 294 0.55 ± 0.09 3576 0.9587 2.3 0.0015
±
0.0001 667 0.9424 5.2
20 45 373 ± 74 0.45 ± 0.04 2380 0.9940 1.1 0.0037
±
0.0006 270 0.9181 8.6
660
661
662
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Table 3. Some quali y pa ame e s be o e and igh a e HPCD ea men o cloudy apple juice a 663
20 MPa and 45 ºC a e 60 min. 664
Pa ame e Be o e HPCD A e HPCD
pH 3.89 ± 0.006
a
3.74 ± 0.006
b
Colo ∆E= 0.51 ± 0.05 L*= 37.30 ± 0.03
a
a*= 1.6 ± 0.1
a
b*= 7.90 ± 0.03
a
L*= 36.98 ± 0.01
b
a*= 1.59 ± 0.08
a
b*= 8.26 ± 0.07
b
To al polyphenols, mg galic/L 456.1 ± 13.1
a
455.4 ± 4.3
a
ABTS, % o inhibi ion
20 min, 49 ± 2
a
60 min, 65 ± 2
a
120 min, 81 ± 3
a
20 min, 52 ± 3
a
60 min, 68 ± 4
a
120 min, 85 ± 4
a
Non-enzima ic b owning,
(mg HMF/L) 2 ± 0.3
a
1.70 ± 0.09
a
PSD D(3,2) = 1.7 ± 0.07 µm
a
D(4,3) = 109 ± 3 µm
a
d(0,1) = 0.59 ± 0.01 µm
a
d(0,5) = 13 ± 1 µm
a
d(0,9) = 337 ± 7 µm
a
Span = 26 ± 7 µm
a
D(3,2) = 0.21 ± 0.01 µm
b
D(4,3) = 2.4 ± 0.2 µm
b
d(0,1) = 0.099 ± 0.001 µm
b
d(0,5) = 0.265 ± 0.006 µm
b
d(0,9) = 1.63 ± 0.07 µm
b
Span = 5.8 ± 0.1 µm
b
Tu bidi y, NTU 105 ± 2
a
168 ± 3
b
Wa e soluble pec in
Oxala e soluble pec in
(mg galac u onic/L)
22 ± 2
a
23 ± 4
a
28 ± 3
a
23 ± 3
a
ξ-po en ial -22.0 ± 0.3
a
-22.3 ± 0.4
a
Values wi h di e en le e s in each ow (a, b) a e signi ican ly di e en when applying he Fishe ’s leas signi ican
665
me hod a p- alue ≤ 0.05.
666
667
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Lis o Figu e cap ions
668
Figu e 1.
(a)
E ec o s i ing speed on PPO inac i a ion om cloudy apple juice by HPCD a 669
20 MPa and 45 ºC (
◇
200 pm, ∆ 400 pm, ○ 600 pm).
(b)
In e se o esidual ac i i y o PPO 670
e sus he p oduc o numbe o e olu ions pe ime and ea men ime (N· ) a 20 MPa and 45 671
ºC (s i ing speed ange: 200 o 600 pm). The con inuous line co esponds o he linea i ing 672
(Eq. 15). 673
Figu e 2.
Inac i a ion o PPO in cloudy apple juice a a mosphe ic p essu e a di e en mild 674
empe a u es (35 ºC
◇
, 40 ºC ○, 45 ºC □). The con inuous lines ep esen he i s o de model 675
(Table 1). 676
Figu e 3.
Inac i a ion o PPO in cloudy apple juice by HPCD
(a)
10 MPa and di e en 677
empe a u es (35 ºC
◇
, 40 ºC ○, 45 ºC □)
(b)
45ºC and di e en p essu es (10 MPa □, 12.5 MPa 678
∆, 15 MPa
◇
, 20 MPa ○). The con inuous lines ep esen he Weibull model (Table 2). 679
Figu e 4
. Inac i a ion o PME in cloudy apple juice unde mild hea ing ea men a 45ºC (∆ 680
a mosphe ic p essu e) and by HPCD a 45 ºC (10 MPa □, 20 MPa ○). The con inuous lines a
681
a mosphe ic p essu e ep esen he i s o de kine ic model (Table 1), while in HPCD ea men 682
ep esen he Weibull model (Table 2). 683
Figu e 5.
Pa icle Size Dis ibu ion (PSD) o cloudy apple juice be o e ea men (
─
) and a e 684
HPCD ea men a 45 ºC, 20 MPa o 60 min (-·-·). 685
686
687
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Figu e 1. (a) E ec o s i ing speed on PPO inac i a ion om cloudy apple juice by HPCD a 20 MPa and 45 ºC
(◇ 200 pm, ∆ 400 pm, ○ 600 pm). (b) In e se o esidual ac i i y o PPO e sus he p oduc o numbe o
e olu ions pe ime and ea men ime (N· ) a 20 MPa and 45 ºC (s i ing speed ange: 200 o 600 pm). The
con inuous line co esponds o he linea i ing (Eq. 15).
0
20
40
60
80
100
0 20 40 60 80
Residual PPO ac i i y, A/Ao·100
ime, min
1
3
5
7
9
11
0 10000 20000 30000 40000
Ao/A
N( pm)·
(b)
(a)
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689
Figu e 2. Inac i a ion o PPO in cloudy apple juice a a mosphe ic p essu e a di e en mild empe a u es (35 ºC ◇,
690
40 ºC ○, 45 ºC □). The con inuous lines ep esen he i s o de model (Table 1).
691
692
693
0
20
40
60
80
100
0 20 40 60 80 100 120
PPO RA, A/Ao·100
ime, min

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694
695
Figu e 3. Inac i a ion o PPO in cloudy apple juice by HPCD (a) 10 MPa and di e en empe a u es (35 ºC ◇,
696
40 ºC ○, 45 ºC □) (b) 45ºC and di e en p essu es (10 MPa □, 12.5 MPa ∆, 15 MPa ◇, 20 MPa ○). The con inuous
697
lines ep esen he Weibull model (Table 2).
698
699
0
20
40
60
80
100
0 20 40 60 80 100 120
PPO RA, A/Ao·100
ime, min
(a)
0
20
40
60
80
100
0 20 40 60 80 100 120
PPO RA, A/Ao·100
ime, min
(b)
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Figu e 4. Inac i a ion o PME in cloudy apple juice unde mild hea ing ea men a 45ºC (∆ a mosphe ic p essu e)
701
and by HPCD a 45 ºC (10 MPa □, 20 MPa ○). The con inuous lines a a mosphe ic p essu e ep esen he i s o de
702
kine ic model (Table 1), while in HPCD ea men ep esen he Weibull model (Table 2).
703
704
705
706
707
708
0
20
40
60
80
100
0 20 40 60 80 100 120
PME RA, A/Ao·100
ime, min
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709
710
Figu e 5. Pa icle Size Dis ibu ion (PSD) o cloudy apple juice be o e ea men ( ─ ) and a e HPCD ea men a
711
45 ºC, 20 MPa o 60 min (-·-·).
712
713
0
1
2
3
4
5
6
7
0.01 0.1 1 10 100 1000
Volume (%)
Pa icle size (µm)